The miR-142 Suppresses U-87 Glioblastoma Cell Growth by Targeting EGFR Oncogenic Signaling Pathway

Glioblastoma is the most lethal malignancy of the brain and is resistant to conventional cancer treatments. Gene-therapy approaches like using tumor suppressor miRNAs are promising in the treatment of glioblastoma. They control the expression of oncogenes and influence tumor features and behaviors. Therefore, in the present study, it was predicted that miR-142 regulates oncogenic epidermal growth factor receptor (EGFR) signaling pathway via TargetScan and miRWalk online tools. Its differential expression level was reduced in glioblastoma according to the previous microarray results, and its predicted target genes were upregulated, as shown by the Expression Atlas. The miR-142 was overexpressed in U-87 glioblastoma cells via lentiviral transduction, and the way it influences proliferation and migration of cells was investigated through MTT assay and wound healing assay. Apoptosis rate was also measured via the Annexin V assay, and cell-cycle analysis was done. Then, real-time polymerase chain reaction (real-time PCR) and western blotting were performed to assess fold changes in mRNA and protein levels of the miR-142 predicted targets. Direct target genes of miR-142 were confirmed through a dual-luciferase reporter assay. The miR-142 significantly suppressed cell proliferation and migration and induced apoptosis and cell-cycle arrest in U-87 glioblastoma cells. This was accompanied by a decrease in expression of SHC adaptor protein 4 (SHC4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), v-akt murine thymoma viral oncogene homolog 1 (AKT1), Kirsten rat sarcoma viral oncogene homolog (KRAS), and mitogen-activated protein kinase 8 (MAPK8) oncogenes at mRNA and protein levels in glioblastoma cells. Also, AKT1 was demonstrated as a direct target of miR-142. Overall, miR-424 acts as tumor suppressor miRNA in glioblastoma cells.


Introduction
Glioblastoma multiform (GBM) is the deadliest malignancy in the central nervous system (1,2). Due to reasons, such as being surrounded by the blood-brain barrier and being so aggressive caused by the mobile nature of glial cells, it has a poor prognosis for conventional treatments of cancer, surgery, chemotherapy, and radiotherapy, with a median survival rate of 9-19 months (3,4).
The miRNAs are small non-coding RNAs, regulating gene expression at the mRNA level via binding to complementary regions in 3' untranslated regions of their target genes (9). They play a crucial role in adjusting many physiological conditions like growth and differentiation and their dysregulation leads to pathological circumstances (10,11). Various candidates of miRNAs are in the potential list of tumor suppressor miRNAs and have been investigated in clinical trials for curing glioblastoma or other types of cancer (12)(13)(14).
In this research, the effect of miR-142 on U-87 glioblastoma cells was studied. For the first time, miR-142 was introduced as a suppressor of several oncogenes of the EGFR signaling pathway in-silico, and then, its role was also checked in-vitro. U-87 cells carry mutations on NF1 and PTEN genes that are inhibitors of Kirsten rat sarcoma viral oncogene homolog (KRAS) and phosphatidylinositol-4,5bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) oncogenes at downstream of EGFR (15). Following these mutations, KRAS and PIK3CA activate their downstream signaling pathways out of control and cause immoderate cell division and survival in glioblastoma cells (7,8). Meanwhile, considering the regulation of these oncogenes through miR-142, inhibition of proliferation and migration of U-87 cells was expected in-vitro.

Experimental
Prediction of the miRNA and target genes TargetScan (www.targetscan.org) and miRWalk 2.0 (zmf.umm.uni-heidelberg.de › mirwalk2) online tools were used to find tumor suppressor miRNAs suppressing several oncogenes of the EGFR signaling pathway (Table 1). After choosing miR-142, its expression was investigated in tumor tissues of patients with GBM versus normal tissues through a microarray dataset (GSE90603) from the Gene Expression Omnibus (www. ncbi.nlm.nih.gov › geo) database. At the next step, expression of miR-142 predicted target Table 1. Predicted target genes of miR-142 in TargetScan and miRWalk databases in common with the EGFR signalin

Cloning of miRNA gene
The miR-142 locus was found in the miRBase (www.mirbase.org) database. The locus was amplified by the polymerase chain reaction (PCR) technique on the genomic DNA template through designing specific primers. The miR-142 amplicon was inserted into the cloning site of the pCDH-GFP-Puro lentiviral vector (System Biosciences) by cutting with EcoRI and BamHI restriction enzymes (Thermo Fisher Scientific).
Cell culture U-87 MG and HEK293T cell lines were provided and fully characterized by the Iranian Biological Resource Center. Both cell lines were cultured in Dulbecco's Modified Eagle's medium (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco) and were incubated at 37 °C with 5% CO 2 and 95% of humidity.

Virus packaging and transduction
For viral packaging, the pCDH vectors containing miR-142 and scrambled were cotransfected to HEK293T cells by psPAX2 and pMD2.G helper vectors (System Biosciences), using poly ethylene imine transfection reagent (Sigma). Cell culture media was changed every 24 h with fresh media, and virus-containing supernatant was collected and stored in the fridge for 4 days. Then, the supernatant was filtered with 0.2 µm sterile filters and was kept at -80 °C freezer. U-87 cells were seeded in an appropriate culture plate with 40% of confluency one day before transduction. Virus-containing supernatants were mixed with fresh media 1:1 and added to cells. To improve transduction efficiency, 10 µg/mL of polybrene (Sigma) was added to cells. The rate of transduction was checked via fluorescent microscopy 48 h after transduction.

MTT proliferation test
Transduced U-87 cells were seeded in a 96-well sterile culture plate at a density of 8 ere seeded in a 96-well sterile culture plate at a density of 8  10 3 ed for 72 h at a 37 °C CO2 incubator. Furthermore, the culture media of fresh media containing 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)romide (Sigma) was added to each well. The plate was incubated for en, the media was removed, and the crystals formed at the bottom of the methyl sulfoxide by shaking and pipetting. Finally, absorbance at 590 te reader (BioTek). re seeded in a 12-well plate at a density of 10 5 cells/well with 80% of a vertical and a horizontal line was drawn on the bottom of each well tip, then the media was removed, and the cell surface was washed with BS). Then, the complete media was added to cells and an image was t 0, 24, 48, and 72 h from the same view of the scratch while culturing. loyed to measure the distance of the cells.
tosis test e seeded in a 24-well sterile culture plate at a density of 410 4 cells/well at a 37 °C CO2 incubator. Then, cells were dissociated with trypsin in ere rinsed twice by a binding buffer of the Annexin V-PE/ 7AAD nces). The cell pellet was dissolved in 200 µL of binding buffer, and E was added to each microtube following incubation for 15 min in the e cells were washed, 5 μL of 7AAD dye was added, and they were y (BD Biosciences). FlowJo 7.6.1 software was employed to analyze e seeded in a 24-well sterile culture plate at a density of 410 4 cells/well at a 37 °C CO2 incubator. Then, the cells were dissociated with trypsin ere rinsed once with PBS. Harvested cells were added to 70% ethanolpwise on vortex and were kept overnight in -20 °C freezer. In the next by centrifuge, and the cell pellet was dissolved in 200 μL of a solution opidium Iodide (Sigma), 1 mg/mL of RNase (Thermo Fisher Scientific), ). Cells were incubated for 40 min in the dark at 37 °C, and then they ometry. FlowJo 7.6.1 software was employed to analyze flow cytometry sing real-time PCR ed using TRIzol (Invitrogen) reagent from the transduced U-87 cells at ufacturer's instructions. For investigation of miRNA expression, stemforward and universal reverse primers, were designed using the method for miR-142 and SNORD47 as an internal control ( Table 2). For n of target genes, random hexamer was used for cDNA synthesis, and 10 3 cells/well and were cultured for 72 h at a 37 °C CO2 incubator. Furthermore, the culture media was removed, and 100 µL of fresh media containing 0.5 mg/ mL of 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (Sigma) was added to each well. The plate was incubated for another 4 h in the dark. Then, the media was removed, and the crystals formed at the bottom of the wells were dissolved in dimethyl sulfoxide by shaking and pipetting. Finally, absorbance at 590 nm was measured by a plate reader (BioTek).

Scratch wound assay
Transduced U-87 cells were seeded in a 12well plate at a density of 10 5 cells/well with 80% of confluency. The next day, a vertical and a horizontal line was drawn on the bottom of each well by a sterile 1-10 µL pipette tip, then the media was removed, and the cell surface was washed with phosphate buffer saline (PBS). Then, the complete media was added to cells and an image was taken by a digital camera at 0, 24, 48, and 72 h from the same view of the scratch while culturing. Image J software was employed to measure the distance of the cells.

Annexin V-PE/ 7AAD apoptosis test
Transduced U-87 cells were seeded in a 24-well sterile culture plate at a density of 4 e seeded in a 96-well sterile culture plate at a density of 8  10 3 for 72 h at a 37 °C CO2 incubator. Furthermore, the culture media f fresh media containing 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)mide (Sigma) was added to each well. The plate was incubated for , the media was removed, and the crystals formed at the bottom of the ethyl sulfoxide by shaking and pipetting. Finally, absorbance at 590 reader (BioTek). seeded in a 12-well plate at a density of 10 5 cells/well with 80% of vertical and a horizontal line was drawn on the bottom of each well ip, then the media was removed, and the cell surface was washed with S). Then, the complete media was added to cells and an image was 0, 24, 48, and 72 h from the same view of the scratch while culturing. yed to measure the distance of the cells.
osis test seeded in a 24-well sterile culture plate at a density of 410 4 cells/well t a 37 °C CO2 incubator. Then, cells were dissociated with trypsin in re rinsed twice by a binding buffer of the Annexin V-PE/ 7AAD ces). The cell pellet was dissolved in 200 µL of binding buffer, and was added to each microtube following incubation for 15 min in the cells were washed, 5 μL of 7AAD dye was added, and they were 10 4 cells/well and were cultured for 72 h at a 37 °C CO2 incubator. Then, cells were dissociated with trypsin in 1.5 mL microtubes and were rinsed twice by a binding buffer of the Annexin V-PE/ 7AAD apoptosis kit (BD Biosciences). The cell pellet was dissolved in 200 µL of binding buffer, and then, 5 µL of Annexin V-PE was added to each microtube following incubation for 15 min in the dark for staining. Next, the cells were washed, 5 μL of 7AAD dye was added, and they were assessed by flow cytometry (BD Biosciences). FlowJo 7.6.1 software was employed to analyze flow cytometry data.

Cell-cycle test
Transduced U-87 cells were seeded in a 24-well sterile culture plate at a density of 4 e seeded in a 96-well sterile culture plate at a density of 8  10 3 for 72 h at a 37 °C CO2 incubator. Furthermore, the culture media f fresh media containing 0.5 mg/mL of 3-(4,5-dimethylthiazol-2-yl)mide (Sigma) was added to each well. The plate was incubated for , the media was removed, and the crystals formed at the bottom of the ethyl sulfoxide by shaking and pipetting. Finally, absorbance at 590 reader (BioTek). a 37 °C CO2 incubator. Then, the cells were dissociated with trypsin in 1.5 mL microtubes and were rinsed once with PBS. Harvested cells were added to 70% ethanol-containing microtubes dropwise on vortex and were kept overnight in -20 °C freezer. In the next day, ethanol was removed by centrifuge, and the cell pellet was dissolved in 200 μL of a solution containing 50 μg/mL of Propidium Iodide (Sigma), 1 mg/mL of RNase (Thermo Fisher Scientific), and Tryton X-100 (Sigma). Cells were incubated for 40 min in the dark at 37 °C, and then they were measured by flow cytometry. FlowJo 7.6.1 software was employed to analyze flow cytometry data.

Gene expression analysis using real-time PCR
The total RNA was extracted using TRIzol (Invitrogen) reagent from the transduced U-87 cells at 72 h, according to the manufacturer's instructions. For investigation of miRNA expression, stem-loop RT primers, as well as forward and universal reverse primers, were designed using the method described previously (16) for miR-142 and SNORD47 as an internal control ( Table 2). For investigating the expression of target genes, random hexamer was used for cDNA synthesis, and specific primers were applied for KRAS, v-akt murine thymoma viral oncogene homolog 1 (AKT1), PIK3CA, SHC adaptor protein 4 SHC4 (SHC4), mitogen-activated protein kinase 8 (MAPK8), and β2 microglobulin (β2M) as an internal control (Table 3). Real-time PCR was done with an ABI StepOnePlus system and SYBR Green Master mix (Ampliqon) under the following condition: 2 min at 95 °C and 40 cycles by 10 s at 95 °C, and 30 s at 62 °C following the melt curve program. The 2 -ΔΔCt method was employed to analyze the data.

Western blotting
The transduced U-87 cells were lysed on ice using radioimmunoprecipitation assay (RIPA) buffer and protease inhibitor cocktail (Merck) at 72 h. After 30 min, the cells' protein extract was isolated by centrifuge, was loaded into a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS (SDS-PAGE) system, and was separated via electrophoresis at the voltage of 150 V. Then, the samples were transferred to a polyvinylidene fluoride (PVDF) membrane, followed by membrane blocking with 5% skim milk (Merck). PBST buffer was used to rinse the membrane. Then, the membrane was incubated in the horseradish peroxidase-conjugated secondary antibody (1:1000; Abcam) solution for 4 h. The enhanced chemiluminescence (ECL) Western Blot Substrate (Thermo Fisher Scientific) was added in a dark room followed by photography. The GelAnalyzer software 2010a was employed to investigate the relative density of bands, and they were normalized with β-Actin.

Dual-luciferase reporter test
The 3' untranslated region of the AKT1

Statistical analysis
All the experiments were performed in triplicate. After analyzing measurements, GraphPad Prism 8 software was employed to calculate mean, and standard deviation among biological repeats, and the Student's t-test was used to define the significance of changes between treatment and control groups, and graphical charts were drawn.

Results of in-silico study suggested downregulation of miR-142 in glioblastoma and upregulation of its predicted targets from the EGFR signaling pathway
For investigating expression changes of miR-142 in patients with GBM, they were checked through available microarray and sequencing data on the GEO database. Analyzing the data from a relevant study (17) (GSE90603 dataset) on 16 glioblastoma tumor samples and 4 standard samples from patients with GBM by GEO2R, it was found that the median differential expression level of miR-142 was equal to 0.687933 in glioblastoma tumor samples compared to 0.852646 in healthy tissue samples ( Figure 1A). So, the median differential expression level of miR-142 was found to be lower in patients with GBM. For studying changes in miR-142 predicted targets in glioblastoma, first, their expression level was checked in sequencing data on the Expression Atlas database. Then, their mutations or expression changes were assessed on the TCGA database. It was found that most miR-142 predicted targets are overexpressed in the glioblastoma state compared to the normal brain state. For most of the genes, the expression level was "High" or "Medium" in the glioblastoma state vs. "Under-cutoff" in normal brain state. Also, it was observed that most of the miR-142 predicted target genes are mutated along with a change in their copy number in glioblastoma, suggesting their role in the progression of glioblastoma tumor (Figures 1B-1C).

The miR-142 inhibited proliferation and migration of U-87 glioblastoma cells
For evaluating miR-142 effects on glioblastoma tumor cells, MTT test and wound healing test were done for U-87 cells at 72 h after transduction of lentiviruses containing miR-142/ scrambled. The rate of transduction was checked through observation of green fluorescent protein (GFP) via fluorescent microscopy ( Figure 2A). Also, real-time PCR was used to show miR-142 overexpression relative to SNORD47 internal control in the transduced U-87 cells. The miR-142 was 58.5 ± 16.3 ( * P < 0.05) folds more expressed in miR-142 transduced cells compared to the scrambled transduced control ( Figure 2B).

The miR-142 induced apoptosis and cellcycle arrest in U-87 glioblastoma cells
The way miR-142 influences apoptosis and cell cycle in glioblastoma U-87 cells was evaluated via flow cytometry. For evaluation of apoptosis, the Annexin V-PE/7AAD apoptosis test kit was used at 72 h after transduction, and the results showed that miR-142 significantly induced apoptosis ( * P < 0.05) in U-87 glioblastoma cells ( Figure 3A-3B). The cell-cycle analysis of U-87 cells was also done via fixing them and PI staining at 72 h after transduction. The flow cytometry results showed a significantly higher number of cells ( * P < 0.05) at the G2 phase with fewer cells at the S phase in miR-142 transduced cells than the scrambled control. In miR-142 transduced cells, 59.9 ± 1.0%, 10.6 ± 0.3%, and 29.4 ± 0.6% of cells were at G0+G1, S, and G2 phases, while in the scrambled transduced cells, 61.1 ± 1.4% of cells were at G0+G1, 14.5 ± 0.6% of cells were at S phase, and 24.9 ± 0.5% of cells were at G2 phase ( Figures 3C-3D). Altogether, miR-142 significantly induced apoptosis and cell-cycle arrest in U-87 glioblastoma cells.

Discussion
The miRNAs are natural oligonucleotides modulating gene expression at the posttranscriptional level. They control physiological processes, such as growth, proliferation, differentiation, and apoptosis by controlling their relevant genes. So, miRNAs have great potential to change the expression of oncogenes in cancer cells and suppress tumor features. Tumor suppressor miRNAs  have been used to inhibit proliferation and migration of glioblastoma cells (18)(19)(20)(21)(22), and some of them are in clinical trial phases (23).
Several previous studies have addressed the tumor suppressor function of miR-142 in glioblastoma (24)(25)(26) or other cancers, such as colorectal (27) and non-small-cell lung carcinoma (28). Qin et al., showed that miR-142 is downregulated in tissues of patients with GBM and its ectopic overexpression in glioblastoma cell lines suppressed cell migration and invasion by targeting Ras-related C3 botulinum toxin substrate 1 (RAC1), leading to suppression of matrix metalloproteinases (MMPs) (24). In a similar study, Li et al., revealed miR-142 downregulation in patients with GBM. They showed that miR-142 overexpression suppresses cell proliferation and induces apoptosis by targeting high mobility group box protein 1 (HMGB1) via the Wnt/β-catenin signaling pathway in glioblastoma cells (25). Consistent with the mentioned studies, our findings approved tumor suppressor role of miR-142 in glioblastoma cells via suppression of cell proliferation and migration and induction of apoptosis and cell cycle arrest and also sheds light on its molecular mechanism of action via regulation of EGFR pathway.
First, miRNA regulators of the EGFR signaling pathway were searched, and it was predicted that miR-142 regulates several oncogenes involved in the EGFR pathway. Most of these oncogenes were overexpressed or mutant in glioblastoma, besides miR-142 was less expressed in glioblastoma tissues vs. normal tissues (Figure 1). EGFR signaling pathway is the most common oncogenic pathway in patients with GBM that causes cell growth (7). Our results showed that predicted target genes of miR-142 from the EGFR pathway, SHC4, KRAS, AKT1, PIK3CA, and MAPK8 become less expressed at mRNA and protein levels by its overexpression via lentiviral transduction. At the same time, proliferation and migration of U-87 glioblastoma cells were reduced, and they were more susceptible to apoptosis and cellcycle arrest in comparison with the control group.
Also, our findings confirmed that AKT1 is a direct target of miR-142 via dual-luciferase reporter assay. When EGFR triggers PIK3CA, it activates AKT1 via phosphorylation. There are various functional proteins at downstream of AKT1. For example, AKT1 activates the oncogenic mammalian target of rapamycin (mTOR) that promotes cell growth and proliferation (29). AKT1 also inhibits apoptosis and promotes survival via functioning on Bad and Bim apoptotic proteins. On the other hand, it was observed that miR-142 downregulated SHC4/KRAS/MAPK8 axis either directly or indirectly. MAPK is another oncogenic module downstream of EGFR that induces cell mobility and migration in glioblastoma cells (30). So, it seemed that miR-142 is a tumor suppressor controlling all these oncogenes.

Conclusion
In conclusion, our findings revealed that miR-142 has therapeutic potential via suppressing glioblastoma cell growth and invasion and can be used as a regulator of oncogenic EGFR signaling pathway as well.

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declaration
The authors declare that there is no conflict of interest.